Revision as of 10:47, 1 April 2013

Contents

General notes

Lots of wild-type inverse pericam plasmid (in the pRSET vector) must be available.

Mutant IPC plasmid (M124S, E67K, and T79P) should also be prepared in advance.

Need to streak out DE3 and DE3/IPC(wild-type) from frozen stocks in advance of liquid culture setup.

To be most careful, may want to freshly transform DE3 with pRSET-IPC miniprep.

DE3 in collection is NB301/AB2

DE3 w/IPC is NB303/AB4

Scheme: each pair of students will make two protein mutants, and test two candidate colonies per mutant. Specifically, students will choose only one mutation of their own, and run a 'positive control' mutation in parallel.

Daily Notes

Enzymes for diagnostics may need to be ordered as well, check designs.

How it went:

Two odd issues cropped up:

In fact, compared to most CaM sequences, two base pairs are missing in the IPC CaM. The Met is easily understandable, the other throws students off more.

Watcut link should be strictly checked and instructions should be included for each pair to name their primer something unique. Sometimes old primers or incomplete enzyme lists were automatically used.

Also, instructions to re-check primer Tm should probably be in bold or even emphasized during pre-lab. I think many folks skipped that step.

Instructor teaches the first group of students how to do the multichannel pipetting (avoid bubbles, make sure all tips are actually taking up the same volume, etc.); then TA takes over while instructor is in BPEC for plate reading.

How it went:

Protein amounts (fluorescence values) were pretty consistently higher this year than in year's past. In part may be due to using Invitrogen instead of Novagen resin, due to a mix-up; in part may be due to the higher cell ODs for many students. Perhaps in future should aim for high-log rather than mid-log growth, in fact.

M124S a much better parallel sample than S101L (run in S09). The change in affinity and cooperativity was dramatic and consistent across the class.

The only strange issue is that at very low calcium concentrations the data is noisy rather than simply being a high plateau, or even somewhat consistently starts a little low, then has the high plateau, then proceeds as expected.